Bottom Line:
These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins.These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors.The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

ABSTRACTMutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Na(v) 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

gks249-F6: RbFox-1 promotes inclusion of SCN8A exon 18A only in the absence of the ESS in a (T)GCATG dependent manner. (A) Scheme of SCN8A wild-type minigenes highlighting the region of the (T)GCATG motifs and subsequent minigenes with RbFox-1-binding sites mutated (B) Agarose gel electrophoresis of cotransfections of the SCN8A WT and SCN8A/ΔESS E18A minigenes without and with the brain specific RbFox-1 expression plasmid. (C) Agarose gel electrophoresis of cotransfections of the SCN8A/ΔESS E18A minigene with mutations in RbFox-1-binding sites, without (CI) and with (CII) brain specific RbFox-1 expression plasmid. Forms of mRNA observed are indicated on the right hand side of the gel. Below analysis of the expression of the RbFox-1 proteins monitored via immunoblotting for the Flag epitope tag present in RbFox-1 expression plasmid and amount of protein loaded on the western blot via immunoblotting against the tubulin. (D) The effect of individual isoforms muscle (m) RbFox-1, RbFox-2 and RbFox-3 proteins in promoting SCN8A exon 18A inclusion analyzed by RT–PCR after cotransfection of overexpression plasmids for these proteins with SCN8A WT and SCN8A/ΔESS E18A minigene constructs. The mRNA species produced are indicated on the right hand side of the gel. On the right side, immunoblot analysis of protein overexpression via antibodies against the flag epitope. Anti tubulin was used as loading control for western blot.

Mentions:
The position and sequence context of a (T)GCATG elements downstream of exon 18A (Figure 6A) has been strongly conserved from Zebrafish to Humans supporting the hypothesis that this element may be a critical component of the splicing switch mechanism that mediates tissue-specific splicing events (47). Furthermore, the (T)GCATG motif is a hallmark of many systems of neuronal splicing regulation (48). The proteins that recognize the hexamers in Humans were found to be homologue of the Caenorhabditis elegans RNA-binding protein feminizing on X known in humans as RbFox-1 family of proteins (49). In mammals there are three RbFox Paralogs: RbFox-1 (A2BP1), RbFox-2 (RBM9) and RbFox-3 (HRNBP3). RbFox-1 is expressed in neurons and muscle cells, RbFox-2 has a broader expression pattern, being observed in stem cells, hematopoetic cells, neurons and muscle. RbFox-3 has only been observed in neurons (43). All paralogs contain a single RNA recognition motif that specifically binds the (U)GCAUG sequence. In addition, the Fox paralogs can be expressed in different isoforms that arise through the use of both alternative promoters and alternative exons (40).Figure 6.

gks249-F6: RbFox-1 promotes inclusion of SCN8A exon 18A only in the absence of the ESS in a (T)GCATG dependent manner. (A) Scheme of SCN8A wild-type minigenes highlighting the region of the (T)GCATG motifs and subsequent minigenes with RbFox-1-binding sites mutated (B) Agarose gel electrophoresis of cotransfections of the SCN8A WT and SCN8A/ΔESS E18A minigenes without and with the brain specific RbFox-1 expression plasmid. (C) Agarose gel electrophoresis of cotransfections of the SCN8A/ΔESS E18A minigene with mutations in RbFox-1-binding sites, without (CI) and with (CII) brain specific RbFox-1 expression plasmid. Forms of mRNA observed are indicated on the right hand side of the gel. Below analysis of the expression of the RbFox-1 proteins monitored via immunoblotting for the Flag epitope tag present in RbFox-1 expression plasmid and amount of protein loaded on the western blot via immunoblotting against the tubulin. (D) The effect of individual isoforms muscle (m) RbFox-1, RbFox-2 and RbFox-3 proteins in promoting SCN8A exon 18A inclusion analyzed by RT–PCR after cotransfection of overexpression plasmids for these proteins with SCN8A WT and SCN8A/ΔESS E18A minigene constructs. The mRNA species produced are indicated on the right hand side of the gel. On the right side, immunoblot analysis of protein overexpression via antibodies against the flag epitope. Anti tubulin was used as loading control for western blot.

Mentions:
The position and sequence context of a (T)GCATG elements downstream of exon 18A (Figure 6A) has been strongly conserved from Zebrafish to Humans supporting the hypothesis that this element may be a critical component of the splicing switch mechanism that mediates tissue-specific splicing events (47). Furthermore, the (T)GCATG motif is a hallmark of many systems of neuronal splicing regulation (48). The proteins that recognize the hexamers in Humans were found to be homologue of the Caenorhabditis elegans RNA-binding protein feminizing on X known in humans as RbFox-1 family of proteins (49). In mammals there are three RbFox Paralogs: RbFox-1 (A2BP1), RbFox-2 (RBM9) and RbFox-3 (HRNBP3). RbFox-1 is expressed in neurons and muscle cells, RbFox-2 has a broader expression pattern, being observed in stem cells, hematopoetic cells, neurons and muscle. RbFox-3 has only been observed in neurons (43). All paralogs contain a single RNA recognition motif that specifically binds the (U)GCAUG sequence. In addition, the Fox paralogs can be expressed in different isoforms that arise through the use of both alternative promoters and alternative exons (40).Figure 6.

Bottom Line:
These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins.These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors.The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

ABSTRACTMutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Na(v) 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.